CD4- and CD8-p56lck binding provided a molecular basis for CD4 and CD8 signaling in T-cells, and has served as a model for the involvement src- related tyrosine kinases in signal transduction. CD4-p56lck complex can signal via the tyrosine kinase domain, and recruit intracellular proteins by means of SH2 and SH3 binding. Recently, we demonstrated lck and fyn SH3 domain binding to the lipid kinase, phosphatidylinositol 3-kinase (PI 3- kinase). In this renewal, we propose to define further the biochemistry of lck and fyn SH3 domain-PI 3-kinase complex and its function in T-cell growth (Aim #I). We propose to define whether there is preferential recruitment of the p85 (alpha,beta) and p110 (alpha,beta) subunits, whether PI 3-kinase binding regulates lck activity and to define the nature PI -P lipids generated by SH3 associated PI 3-kinases. Preliminary data indicates that PI 3-kinase is required for DNA synthesis, but not interleukin 2 production. We propose to define the role of SH3-PI 3-kinase binding in TcR-CD4 induced proliferation by transfection with the isolated p85 subunit. In the second part of the application, we present exciting new data that a second intracellular protein, PI 4-kinase, can associate directly with the cytoplasmic tails of CD4 and CD8. Further, it binds to a site distinct from the p56lck binding site. Although CD4 and CD8 co- receptor function depends on p56lck binding, thymic differentiation has been reported to occur independent of associated p56lck. PI 4-kinase is an excellent candidate to regulate these events. In Aim #2, we propose to map the site of binding within the CD4 and CD8 antigens and to determine the identity of the PI 4-kinase. We then propose to reconstitute of PI 4- kinase binding loss mutants in CD8 or CD4 deficient mice in order to determine its potential role in positive/negative selection in the thymus.